The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art.
Until recently, a persistent dogma of neuroscience was that neurons in the adult human brain and spinal cord could not regenerate. However, in the mid-1990s, neuroscientists learned that some parts of the adult human brain do, in fact, generate new neurons. These new neurons arise from “neural stem cells” in the fetal as well as the adult brain. The discovery of a regenerative capacity in the adult central nervous system holds out promise that it may eventually be possible to repair damage from neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), as well as from brain and spinal cord injuries resulting from stroke or trauma. However, the non-invasive isolation and purification of significant numbers of neural stem cells from the brain remain challenging.
On the other hand, embryonic stem (ES) cells are pluripotent cells that are both self-renewing and have the capacity to differentiate into any cell type in the human organism. They can be propagated in vitro for long periods of time in an undifferentiated state and thus represent an attractive source of cells for developmental studies and for therapy of human diseases.
ES cells are responsive to environmental cues upon transplantation and adopt a cellular fate that is appropriate to the transplanted region. Nevertheless, the dependence on environmental cues to direct stem cells precludes the efficient generation of neurons in non-neurogenic regions of the CNS.
Recently, it has become possible to direct the differentiation of stem cells ex vivo, theoretically making these cells less dependent or independent of in vivo cues. For example, spinal motorneurons can be generated efficiently by exposing mouse ES cells to retinoic acid (RA) and Sonic Hedgehog. In this paradigm, RA serves both to neuralize and to establish a caudal positional identity for the pluripotent ES cells. Sonic hedgehog or Hedgehog agonist (HhAg1.3) further specifies a ventral positional identity and in response, a substantial proportion of ES cells initiate a motor neuron-specific transcriptional pattern and acquire immunohistochemical features of mature neurons. ES cell-derived motoneurons transplanted into embryonic chick spinal cord extend axons into the periphery and form neuromuscular junctions. [Wichterle, H., Lieberam, I., Porter, J. A. & Jessel, T. M. Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385-397 (2002)].
The therapeutic potential of embryonic stem (ES) cells is promising, but in many cases limited by our inability to promote differentiation to specific cell types, such as motor neurons. Accordingly, there is a pressing need for technology to generate more homogenous differentiated cell populations from pluripotent stem cells.
The isolation and characterization of two motoneuronotrophic factors (MNTF1 and MNTF2) from rat muscle tissues as well as the subsequent cloning of a recombinant MNTF1-F6 gene derived from a human retinoblastoma cDNA library, is described in U.S. Pat. Nos. 6,309,877, 6,759,389 and 6,841,531 (as well as co-pending U.S. patent application Ser. Nos. 10/858,144, 10/858,286, 10/858,543 and 10/858,545). Nucleotide sequences encoding polypeptides related to MNTF1, were found to map within human chromosome 16q22, as described in International Application No. PCT/US2004/038651.
The MNTF1-F6 gene sequence encodes a 33 amino acid sequence. The naturally occurring and recombinant MNTF1 polypeptides were shown to selectively enhance the survival in vitro of anterior horn motor neurons isolated from rat lumbar spinal cord explants. Photomicrographs of treated cultures exhibited neurite outgrowth of myelinated nerve fibers and a marked reduction in the growth of non-neuronal cells, e.g. glial cells and fibroblasts. Similarly, in vivo administration of MNTF1 to surgically axotomized rat peripheral nerves resulted in a markedly higher percentage of surviving motor neurons than untreated controls, which could be blocked by co-administration of anti-MNTF1 monoclonal antibody.
Further beneficial effects of MNTF1 were demonstrated in rats subjected to spinal cord hemi-section, repaired by a peripheral nerve autograft and implanted with MNTF1-containing gel sections in close proximity to the nerve graft junctions with spinal cord. MNTF1 treated animals exhibited greater numbers of surviving motor neurons, improved recovery of motor and sensory function, reduced inflammatory response (fewer infiltrating macrophages and lymphocytes) and reduced collagen-containing scar tissue formation at the site of the graft, normal Schwann cell morphology and normal myelinated and non-myelinated nerve fiber formation.
The efficacy of MNTF in the treatment of neurodegenerative disease was also demonstrated in the wobbler mouse animal model. Wobbler mice carry an autosomal double-recessive gene mutation that leads to the progressive degeneration of spinal and brain stem motor neurons. Implantation of MNTF1-containing gel sections between the trapezius and rhomboid muscles and the C7-T3 region of the spinal cord delayed the progression of symptoms in wobbler mice, resulting in a general improvement in life span, health, respiration, body weight, strength of forelimbs as well as reduced vacuolation and chromatolysis of their cervical motor neurons compared to the control group.
Two overlapping domains within the MNTF1-F6 molecule that appear to be sufficient for the known biological activities of MNTF1 were identified. See, International Application No. PCT/US04/01468 or U.S. patent application Ser. No. 10/541,343. Each of these domains, designated herein as the “WMLSAFS” and “FSRYAR” domains, were sufficient to stimulate the proliferation of motor neuron derived cell lines in a manner similar to the MNTF1-F6 33-mer. Similarly, the “FSRYAR” domain is sufficient to direct selective reinnervation of muscle targets by motor neurons in vivo in a manner similar to the MNTF1-F6 33-mer. In addition, the “FSRYAR” domain provides an antigenic epitope sufficient to raise antibody that recognizes any MNTF peptide containing the “FSRYAR” sequence, including the MNTF1-F6 33-mer.
Clearly, more efficient and selective methods are needed to direct the proliferation and the differentiation of stem cells, especially ES cells, to produce homogenous populations of motor neurons. This may be important not only for the therapeutic use of stem cells in the treatment of neurodegenerative disorders, but will also greatly facilitate studies of the molecular mechanism of development.